Problems using NexteraXT with mitochondrial DNA

[MicroBio]

Hi all,

I just prepared a NexteraXT library of mitochondrial DNA for a collaborator. Although they purified the mitochondrial DNA (removed genomic DNA), the sequencing results from the MiSeq suggest that it is still almost completely genomic DNA contaminants.

Does anybody have experience performing shotgun sequencing with mitochondrial DNA or, even better, using NexteraXT with the mito DNA? I figure one possibility is that the NexteraXT is not able to properly 'tagment' the mitochondrial DNA and is only processing the genomic DNA. The other possibility is that the samples are not pure enough.

If anybody could offer insight into preparing mitochondrial DNA libraries with NexteraXT, that would be amazing!

[JackieBadger]

We use Nextera to sequence whole mtDNA genomes. We have no mtDNA purification step and get good coverage across the whole genome no worries (not the most efficient way as most of the data is genomic DNA). We have included up to 11 samples of varying species with decent size genomes and recovered all mtDNA genomes.

[MicroBio]

Thanks JackieBadger! What kind of coverage do you get on the mtDNA genomes? Also, you are using Nextera and not Nextera XT right (although my understanding is that they are pretty similar)?

[JackieBadger]

I think just Nextera...I would have to check with the lab tech. We get a mean coverage of around 10x I think

[MicroBio]

Okay cool! Is that on the MiSeq or HiSeq?

[ScottC]

Hi MicroBio,

We have sequenced many mito genomes using NexteraXT and the MiSeq. They should work well together.

[MicroBio]

Thanks ScottC!

[dnatimelord]

Scott, are you retrieving whole mtDNA genomes from genomic DNA just by shotgun sequencing or are you doing medium-long range PCRs for mtDNA first?

[concitacantarella]

Hi all,
we sequenced mitochondrial plant genomes using Nextera kit and MiSeq machine.
We also purified the mitochondrial DNA.
The analysis still highlights a great nuclear contamination ( about 80%) and, above all, an odd fragment size distribution.
The insert size distribution isn't Gaussian, the higher point is about 280nts while the library size was about 860 bp.

Has anyone sequenced mitochondrial plant genomes( 450 kbp), from purified sample, with Nextera kit?

[nucacidhunter]

Nuclear contamination is indicator of purification method efficiency. The fragment size distribution for Nextera library prep methods is wide (100-2000 bp) without post-PCR clean up. The peak will also look different depending on the instrument and type of Chip used. For instance, using DNA 1000 Chip is less likely to reveal the whole distribution range of fragments or the real peak. If you are using MiSeq long reads (2x250 or 2x300 cycles) it would be better to clean PCR product with 0.5 x beads to remove majority of smaller fragments (<500 bp) to better utilise the long reads. A peak of 280 nts insert for your sequenced fragments in comparison with input library peak of 860 indicates that post-PCR clean-up was not stringent and lots of smaller fragments were carried over. Because smaller fragments are more efficient in cluster formation there is a large shift in fragment size of reads in comparison to input.

If mitochondrial genome can be purified effectively with less of genomic DNA, Nextera XT with 1<= ng of input would provide better results.

[concitacantarella]

Thanks a lot for suggestions.
Comparing the insert-size distributions after nuclear and mitochondrial alignment, we identify the subset of reads which preferentially aligns the mitochondrial reference. This subset has size > 500bp.
Probably we have to better clean the PCR product with the beads concentration which you suggest.

[rnaeye]

Hi! concitacantarella,
I have tried NEB's NEBNext Microbiome DNA Enrichment Kit. It also enriches for mitochondrial DNA since methylation is very low or absent in mito genome.